Project Summary: This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 20-012. The goal of this supplement is to build upon the amazing progress made in the parent Phase 1 SBIR in developing a less immunogenic asparaginase (ASNase) by adding pivotal studies that would demonstrate its reduced immunogenicity, potent anti-ALL action, and superior safety profile. ASNases are enzyme drugs that systemically deplete asparagine from the blood and are a mainstay of treatment for acute lymphoblastic leukemia (ALL), a cancer of white blood cells and the most common pediatric cancer. Despite being highly effective in the treatment of pediatric ALL, ASNases are associated with a multitude of toxic side effects, some so severe as to be fatal. Their high toxicity precludes routine treatment in adult ALL, contributes to a much lower cure rate of <40% for adults, and prevents their use in other hematological malignancies (e.g. acute myeloid leukemia) and solid tumors (e.g. pancreatic cancer), despite strong preclinical efficacy evidence. The two main sources of toxicity are L-glutaminase (GLNase) co-activity and immunogenicity due to their bacterial origins. Since the toxic side effects can prevent or terminate treatment, there is a clear unmet clinical need for an safer ASNase with reduced immunogenicity and without GLNase co-activity. We are currently developing an ASNase that addresses both problems - it is devoid of GLNase co-activity and mammalian in origin, specifically guinea pig (GpA). We truncated and further humanized GpA to further reduce the risk of immunogenicity. In our active Phase 1 SBIR, we identified potential antigenic epitopes and engineered deimmunized variants (GpA369hum-DI; >84% identity to the human homolog compared to ~25% identity for current ASNases), whose lessened ability to stimulate a T cell response will be confirmed in the grant?s last two months. While clinical immunogenicity is extremely complex and impacted by many intrinsic and extrinsic factors, we are mitigating this risk by assessing potential immunogenicity issues at this early preclinical stage to avoid possible failure in the clinic, where costs are much higher, and patients? lives are at risk. This supplemental grant will enable us to demonstrate reduced immunogenicity caused by cytokine release syndrome (Aim 3) as well as provide critical preclinical evidence of in vivo bioavailability and efficacy of GpA369hum-DI in both B- and T-cell ALL (Aim 1) and a superior safety profile in toxicology studies (Aim 2). This will strengthen our Phase 2 SBIR application by establishing preclinical feasibility, de-risking our lead candidate before entering clinical trials, and provide evidence of its value propositions to enhance its attractiveness for commercialization to private investors.